1

Metabolomics and Mammalian Cell Culture

Kathya De la Luz-Hdez
Center of Molecular Immunology
Cuba

1. Introduction
Since the mid-1950s, when pioneering work of Earle and colleagues (1954) enable routine
cell culture, mammalian cell culture has been used in the large-scale production of
recombinant protein and monoclonal antibodies. Mammalian cell lines are preferred as
production host for many pharmaceuticals, since complex post-translational modifications
of the produced proteins (especially glycosylation) are generally not properly performed by
microbial systems (Lake-Ee Quek et al., 2010).
Wagburg described that under batch conditions, mammalian cells display an inefficient
metabolic phenotype characterized by high rates of glucose to lactate conversion (Warburg,
1956) together with partial oxidation of glutamine to ammonia and non-essential amino
acids (Fitzpatrick et al., 1993; Jenkins et al., 1992; Ljunggren and Haggstrom, 1992; Ozturk
and Palsson, 1991). The accumulation of fermentation by-products causes a reduction of the
culture density and product titer that can be realized (Martinelle et al., 1998).
In order to increase the cell productivity a common optimization approach is to grow cells
to moderately high density in fed-batch and the deliberately induce a prolonged, productive
stationary phase. While optimization of this perturbed batch strategy is responsible for the
increase of monoclonal antibodies titer seen over the past decades it has a number of short-
comings, including:
a. the strategy has to be refined for each new cell line,
b. the ultimate metabolic phenotype during prolonged stationary phase varies between
cell lines and it is not always possible to achieve the most productive phenotypes for a
given strain
c. volumetric productivity remains relatively low due to moderate cell density (Lake Ee
Quek et al., 2010).
Other approaches are related with the changes of cell phenotypes through metabolic
engineering or change of culture media conditions in order to manipulate the cellular
metabolic behavior.
Although transcriptomics and proteomics have been explored extensively for mammalian
cell engineering (Korke et al., 2002; Seow et al., 2001; Seth et al., 2007; Smales et al., 2004; de
la Luz et al., 2007, 2008) these tools fall short of generating direct measurements of the
physiological state of the cell. It is essential to combine these techniques with metabolic flux

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4 Metabolomics

analysis (MFA) a powerful method to quantify the manifestation of a phenotype: the

intracellular reaction rates or the fluxome. One of the relatively new omic sciences is the
field of metabolomics. The metabolome was first described by Oliver and collagues (1998) as
being the set of all of low-molecular-mass compounds synthesized by an organism.
Metabolomics is therefore the analysis of small molecules that constitute the metabolism,
and it offers the closest direct measurements of a cells physiological activity (Beecher, 2002;
Khoo and Al-Rubeai, 2007). The metabolomic analysis can be considered as the
measurement of the change in the relative concentrations of metabolites as the result of the
deletion or overexpression of a gene, should allow the target of a novel gene product to be
located on the metabolic map. Another definition of the metabolome states that it consists
of only those native small molecules that are participants in general metabolic reactions
and that are required for the maintenance, growth and normal function of a cell (Khoo and
Al-Rubeai, 2007).
The metabolomic as a new powerful tool to understand the complex processes of large scale
mammalian cell cultures for biopharmaceutical production has not been yet embraced during
process development and scale-up. This is mostly because metabolites are now not the
primary focus and the relationship between metabolites and protein production in different
media are not fully understood. It is for this reason that metabolomics can bridge the gap of
understanding as to the dynamics of metabolism, cell growth and protein production (Khoo
and Al-Rubeai, 2007). The metabolomics can be use to optimize conditions of bioreactors or the
development chemically defined media. Characterizing cell lines, culture media and selection
of cell lines are a vital step in the process development of biologics. In this chapter, we describe
the state-of-the-art of the use of metabolomics tool in mammalian cell lines.

2. Complexity of metabolome analysis

Metabolomics requires the unbiased identification and quantification of all of the
metabolites present in a specific biological sample (from an organism or in vitro).
Metabolites are generally labile species, by their nature are chemically very diverse, and
often present in a wide dynamic range. For analysis of mRNA and proteins one only
needs to know the genome sequence of the organism and exploit this information using
nucleic acid hybridization or protein separation followed by MS (although PTM are
problematic). However, the analysis of metabolites is not as straightforward. In contrast to
transcripts or protein identification, metabolites are not organism specific (that is to say,
sequence dependent) (Hollywood et al., 2006).
In addition, their diverse chemical properties make complete metabolite analysis difficult.
Genes are composed of a linear four-letter code, whereas proteins have a 20-letters code of
primary amino acids. Metabolites do not have any fixed codes, and thus a general method of
characterization is difficult. Present methods use the specific chemical properties of these
entities to separate, identify and decipher their structures. Combinatorial approaches allow
for a greater coverage. An ideal metabolic analysis should provide:
a. Give an instantaneous snapshot of all metabolites in any given system,
b. Use analytical methods that have high recovery, experimental robustness,
reproducibility, high resolving power and high sensitivity (Fiehn, 2001) whilst being
able to be applied universally,

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Metabolomics and Mammalian Cell Culture 5

c. Provide the unambiguous quantification and identification of metabolites and

(d) factors to be highlighted while easily being incorporated into biochemical network
models (Soo H and Al-Rubeai M, 2007).
Before any metabolome measurements are taken it is essential that metabolism is stopped as
quickly as possible, especially because the enzymes are active. For animal cells liquid N2 is
used to snap freeze the sample, followed by mechanical disruption in order to release the
metabolites (Viant et al., 2005). The next stage of the analysis is to extract the metabolites.
There are many different methods (Tweeddale et al., 1998; Buchholz et al., 2001; Villas-Boas
et al., 2005) and the most common ones are:
- Acid extraction using perchloric acid, followed by freeze thawing, then neutralization
with potassium hydroxide
- Alkali extraction typically using sodium hydroxide, followed by heating (80oC)
- Ethanolic extraction by boiling the sampling in ethanol at 80oC
When the extract is finally ready, the choice of the analytical tool is based on the level of
chemical information required about the metabolites, remembering that there will be a
chemical bias with respect to that method, and the speed of analysis is also another
consideration. The figure 1 shows different methods and approaches used for the
metabolomic analysis.

Fig. 1. Technologies for metabolome analysis. GC-MS: gas chromatography mass

spectrometry, GCxGC-MS: 2 dimensional GC coupled to mass spectrometry, LC-MS: liquid
chromatography mass spectrometry, HPLC: high performance liquid chromatography, LC-
NMR: liquid chromatography coupled to nuclear magnetic resonance, NMR: nuclear
magnetic resonance, LDI-MS: laser desorption ionization mass spectrometry, FT-IR: Fourier
transform infrared spectroscopy

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6 Metabolomics

The most popular approaches are:

1. Metabolite target analysis: which is an approach that is restricted to metabolites for
example a particular enzyme system that would be directly affected by any
perturbation
2. Metabolite profiling: which is focused on a specific group of metabolites (for example
lipids) or those associated with a specific pathway; within clinical and pharmaceutical
analysis, this is often called metabolic profiling, which is used to trace the fate of a drug
or metabolite
3. Metabolomics: is the comprehensive analysis of the entire metabolome, under a given
set of conditions
4. Metabonomics: which seeks to measure the fingerprint of biochemical perturbations
caused by disease, drug and toxins.
5. Metabolic fingerprinting: is used to classify samples based on provenance of either their
biological relevance or origin by using a fingerprinting technology that is rapid but
does not necessarily give specific metabolite information.

3. Experimental design
3.1 Cell culture growth, stimulation
The differences in optimized cell culture growth conditions present another major concern
for cell line metabolomics. This is particularly an issue is studies involving comparative
analysis of several different cell types, all of which might contain different levels of glucose,
glutamine and lactate, as well as other nutrients and additives, which will probably lead to
differences in the metabolome of the cells. If possible, it is recommended to use the same
growth medium for all cell lines in the study to reduce variance in metabolic profile that can
be caused by the medium (Cuperlovic-Culf et al., 2010). The standard enhancement of cell
culture medium with serum of animal origin can add another level of complexity in cell
growth condition optimization. Variations in serum can lead to contamination with
exogenous metabolites and alterations of endogenous cell metabolite.
In order to minimize the influence of different cell culture conditions in the metabolomic
final results, proper experimental designs are crucial. Nonetheless, more effort is required in
the future for the determination of metabolic differences caused by various growth
conditions, cell culture age and/or passage number for different cell lines.

3.2 Sample preparation and metabolite extraction

The goal of metabolomics is to analyze all or, at least, as many as possible different
metabolites without selectivity for any particular molecular type and/or characteristics. The
correct sample preparation is the first step in order to ensure the detection of a large number
of metabolites. As metabolic processes may be rapid, varying from milliseconds to minutes
(Gerdtzen et al., 2004; Taoka and Banerjee, 2002), the first necessary step is to rapidly stop
any inherent enzymatic activity or any changes in the metabolite levels. The time and
method of sampling are important issues to be considered to ensure reproducibility in the
analytical sample, especially since a large number of biological replicates is commonly used
(Khoo and Al-Rubeai, 2007).

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Metabolomics and Mammalian Cell Culture 7

These methods include freeze-clamping (with lower-temperature receptacles), immediate

freezing in liquid nitrogen or by acidic treatments (ap Rees and Hill, 1994). Freezing in
liquid nitrogen is generally considered to be the easiest way of stopping enzyme activity
provided that cells or tissues are not allowed to partially thaw before extracting metabolites.
In order to prevent this from happening, enzyme activity is inhibited by freezing-drying or
by immediate addition of organic solvents while applying heat. Cells are subsequently
disrupted, releasing the metabolites. Frozen samples may be ground down by sonication,
homogenization by mechanical means in pre-chilled holders (Fiehn et al., 2000) or directly in
an extraction solvent (Orth et al., 1999). The mixture of cell debris, protein, nucleotides and
the desired metabolites need to be separated; this can be done by centrifugation or filtration.
For the complete analysis of a cell culture, it is important to measure both extracellular
(footprint) and intracellular (fingerprint) metabolic profiles. Metabolic footprinting is
technically simple because it requires only centrifugation to separate culture media and cells
before the analysis. Metabolic fingerprinting, although much more technically challenging
because it requires metabolite extraction from cells, provides more complete information about
cellular metabolic processes (Cuperlovic-Culf et al., 2010). Recently a study related with
different metabolite extraction protocols for mammalian cell culture was published (Dietmair
et al., 2010). In this study, the authors compared 12 different extraction methods, according to
their results; extraction in cold 50% aqueous acetonitrile was superior to other methods.

3.3 Analytical Instruments platforms

Currently, the main analytical techniques used for the analysis of the metabolome are
nuclear magnetic resonance spectroscopy (NMR) and hyphenated techniques such as gas
chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS).
In addition other combinations are possible, e.g. capillary electrophoresis (CE) coupled to
MS or LC coupled to electrochemical detection. Alternatively, Fourier transform infrared
spectroscopy (FTIR) and direct infusion mass spectrometry (DIMS) have been applied
(Dunn and Ellis, 2005; van Greef et al., 2004; Lindon et al., 2007; Koek et al., 2010) without
any prior separation, except for eventual sample preparation. NMR, FTIR and DIMS are
high throughput methods and require minimal sample preparation and may be preferred
techniques for metabolic fingerprint. However, the obtained spectra are composed of the
signals of very many metabolites and elucidation of these complex spectra can be very
complicated. In addition, detection limits for NMR and FTIR are much higher that for MS-
based techniques, limiting the application range to metabolites present in higher
concentrations. Therefore, GC, LC and CE coupled to MS are generally preferred in
metabolomics to allow quantification and identification of as many as possible metabolites.
The general requirements for metabolomic instruments are:
- Excellent sensitivity and resolution for a wide range of molecules types
- The ability to handle a large range of concentrations (from pM to mM) for different
molecular types
- The ability to identify and quantify different molecules
- Short analysis time
- To enable the measurement of many samples without sample degradation during the
measurement
- Reproducible measurement across different centers and time

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8 Metabolomics

Several reviews have dealt with the application of NMR and MS in metabolomics (Ala-
Korpela, 2008; Detmer et al., 2007; Griffin, 2003). NMR is a non-invasive, non-destructive,
highly discriminatory and fast method that can analyze rather crude samples. NMR
spectroscopy can be performed without extensive sample preprocessing and separation and
provides several different experimental protocols optimized for mixture analysis and
molecular formula or structure determination. The results of NMR measurements have
proven highly replicable across centers and instruments (Viant et al., 2005). NMR can
provide measurements for different types and sizes of both polar and non-polar molecules
through analysis of different spectral windows. In addition, NMR instruments are highly
versatile and with only minor changes in probes, users can obtain spectral information for
different nuclei (1H, 13C, 15N, and 32P among others) in solvent or solid samples and even in
vivo (Griffin, 2003). NMR is also the only method used in metabolomics that currently
enables direct measurements of molecular diffusion, interactions and chemical exchange.
Several databases and methods are being developed that enable metabolite identification
and quantification from NMR spectra (Table 1). The major problem with NMR technology
as applied to metabolomics is its low sensitivity, which limits the majority of currently
available instruments to measurement of fewer than 100 metabolites.
The role of MS in metabolomic research is constantly expanding, whether the focus is on
profiling (targeted analysis) or pattern-based analysis (Hollywood et al., 2006). Recent
technological advances in separation science, ion sources and mass analyzers have
considerably increased the sensitivity, selectivity, specificity and speed of metabolite
detection and identification by MS. There are five important considerations that need to be
dealt with in any global metabolite analysis by MS:
1. The efficient and unbiased extraction of metabolites from the sample matrix
2. Separation or fractionation of the analytes by chromatography
3. Ionization of the analyte metabolite
4. Detection of mass signals
5. Analyte identification

Name and availability Instrument Additional information

Human Metabolome Project Biological data; chemical and
NMR, MS
(http:/www.hmdb.ca) clinical data specific to humans
Database search for NMR peaks
BMRD (http:/www.bmrb.wisc.edu) NMR
assignment
Prime (http:/prime.psc.riken.jp) MS, NMR
Glom metabolome database
MS Specific to plants
(http:/csbdb.mpimp-glom.mpg.de)
METLIN metabolite database Drug and drug metabolites;
MS
(http:/metlin.scrpps.edu) specific to humans
NIST chemistry WebBook
NMR, MS, IR
(http:/WebBook.nist.gov/chemistry)
Madison metabolomics database
MS, NMR
(http:/mmcd.nmrfarm.wisc.edu)
NMR Lab of biomolecules Databse search for NMR peaks
NMR
(http:/spinportal.magnet.fsu.edu) assigment

Table 1. Databases of metabolomic standard data for quantification and assignment

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Metabolomics and Mammalian Cell Culture 9

Separation of analytes before MS detection is an important step leading to detection of

more features, effectively increasing the overall peak capacity of the analytical
platform. Separation methods include condensed-phase separation methods and gas-
phase analyte separation. DIMS relies solely on the mass spectrometer to perform
separation and offers an advantage in terms of speed and sample throughput. The
number of identified features in a MS measurements can also be increased by changing
the polarity of the ion source. Positive ion mode electrospray is optimal for basic
metabolites (e.g. amines). Negative ion mode provides optimal measurement for acidic
metabolites. Knowledge of the empirical formula based on exact mass can often be used
to assign one or a few putative identifications that can then be used for searching
metabolic or chemical databases (Table 1).
A comparative outline of the characteristics of NMR and MS methodologies as applied to
metabolomics is provided in Table 2. The two methods are highly compatible and, thus
ideal approach is to combine the results from NMR and MS measurements.
The most common forms of chromatography are GC and LC. GC runs are relative long, at
about 60 min or more (Gummer et al., 2009); however, deconvolution software allows for
the decrease in run times. In LC there is a shift from standard HPLC to UPLC (ultra-
performance liquid chromatography), which can significantly increase resolution sensitivity
and peak capacity (Gummer et al., 2009) due to the reduced particle size, while decreasing
sample volumes and mobile phases. UPLC systems operate at high operating pressures and
use sub-2-m porous packing. Unlike pressured systems such as LC, CE (capillary
electrophoresis) makes use of an electric field to move molecules towards the detector, much
like gel electrophoresis. CE coupled with UV or LIF (laser-induced fluorescence) detectors
are highly sensitive, but lack selectivity.
GC coupled to MS is one of the most common instrument platforms to be used in
metabolomics experiments. GC-MS instruments using linear quadrupole analysers have
been available for decades providing a robust technology that is amenable to
automation. The identification of a wide range of primary metabolites (often after
derivitisation) is greatly facilitated by the high resolution of capillary GC, the
reproducible fragmentation of metabolites in the mass spectrometer and the ready
availability of large mass spectral libraries (Roessner et al., 2001). Recent developments
in GC-MS have resulted in improvements in both the GC and MS capabilities of this
platform and a move towards the use of high mass accuracy/ high mass resolution
instruments. The requirement for high throughput has led to the use of nominal GC-
time-of-flight TOF-MS with much faster scan rates. High scan rates allow rapid
temperature gradient programs, resulting in shorter run times and increased sensitivity
(Gummer et al., 2009). Alternatively, the combination of two-dimensional GC with TOF-
MS has resulted in the development of very high resolution fast MS that can be used to
detect more metabolites than is possible using single queadrupole (Q), TOF and ion trap
mass analyzer.
One if the drawbacks of many GC-MS metabolomics analyses is the need to derivitise
metabolites before analysis. In contrast, many classes of polar metabolites can be analyzed
directly by LC-MS without derivitisation. LC systems interfaced with TOF mass analyzer
are now commonly used in metabolomics analyzes, delivering high throughput and high

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10 Metabolomics

mass resolution analysis capability with mass accuracy approaching single digit ppm.
Recently developed instruments also allow rapid polarity switching between positive and
negative mode within a single run, reducing the need for multiple runs and cost per sample
(Gummer et al., 2009).

Analysis NMR MS
High throughput-metabolites No Medium
High throughput-samples; automation Yes No
Quantitative Yes Yes
Availability in clinic No No
Equipment cost High High
Maintenance cost Medium High
Per sample cost Low High
Required technical skills Yes Yes
Sensitivity Medium High
Reproducibility High Low
Data analysis automation Yes Yes
Identification of new metabolites Difficult Possible
Chemical exchange analysis Yes No
In vivo measueremnt Possible Impossible
Table 2. Comparison of characteristics of major experimental methods for metabolomic
analysis

LC-MS linear quadrupole, triple quadrupole (QQQ), QTrap and io trap mass analyzers have
also been utilized for global and targete metabolomics, but may be limited by mass accuracy
and mass resolution in identifying metabolites. However, the use of triple quadrupole and
QTrap mass analyzers in various selective ion scaning modes can be used to detect specific
metabolites or metabolite classes with high sensitivity and are particulary useful for targeted
metabolomic analysis.
CE-MS offers a complementary approach to LC-MS for analyzing anions, cations, and
neutral particles in a single run. Metabolites can be analyzed directly without derivitisation
and the chromatographic resolution and sensitivity of CE is very high. However, CE is less
frequently used for metabolomic analyzes tha LC-MS.
Vibrational spectroscopies are relative insensitive, but FTIR allows for high throughput
screening of biological samples in an unbiased fashion. Similar to NMR, water signals pose a
problem and must be subtracted electronically or attenuated total reflectance may be used.
Compared with the other methods it is one of the least sensitive, but its unbiasness to
compounds and ability to analyse large numbers of sample in a day makes it a plausible
method for screening purposes (Khoo and Al-Rubeai, 2007).
LC-MS-based instruments can be operated in direct infusion mode with no chromatographic
separation for measurement of the total mass spectrum for the mixture. The infusion can be
performed with either the LC autosampler or with and offline syringe pump. Ion trap, TOF,
Q-TOF, Orbitrap and FT-ICR-MS mass analyzers have been used with this mode of sample
delivery. This approach relies totally on the mass analyser to resolve isobaric metabolites
such as leucine or isoleucine. The key advantange of direct infusion analysis is the potential

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Metabolomics and Mammalian Cell Culture 11

for automated high throughput sample analysis with both low and high mass resolution
mass analyzers.
Another beneficial experimental method for cell culture metabolomics analysis involves
stable isotope labeling followed by either MS or NMR measurement. This approach enables
pathway tracing, easier metabolite assignment and metabolic flux measurements. Isotopic
labeling has previously enabled detailed determination of pathways leading to the
production of specific metabolites and the development of the highly accurate mathematical
models of these pathways (Hollywood et al., 2006).

4. Aplications in mammalian cell culture Study cases

4.1 Analysis of molecular mechanisms associated to the adaptation of NS0 myeloma
cell line to protein-free medium
The NS0 mouse myeloma cell line has become one of the most popular systems for large-
scale heterologous protein expression. For reasons of regulatory compliance, cost, batch
consistency, downstream processing, and material availability, industrial applications of
NS0 has moved towards serum or protein-free medium platforms (Barnes et al., 2000). For
serum- or protein-free cultivation, the cell culture medium is often supplemented with
lipids (derived from plant or synthetic sources) in addition to other protein supplements.
The effect of lipid supplementation on the physiology of hybridomas and myelomas has
been reported (Jenkins et al., 1992). NS0 cells are naturally cholesterol-dependent; not only is
their growth greatly facilitated by lipid supplementation, but is also dependent on provision
of cholesterol. NS0 cells capable of cholesterol-free growth can be isolated by selecting
mutant clones or by adaptation. Adaptation generally involves passaging cells over a time
period during which the serum concentration is decreased gradually (Sinacore et al., 2000).
Eventually, the resulting population develops the capability to grow in the absence of
serum. Different mechanisms underlying a cholesterol-dependent phenotype could include
the absence (or mutation) of a gene or a segment of gene along the cholesterol biosynthesis
pathway. There could be changes in the expression level of some proteins of the pathway
due to gene regulation or other control mechanisms. In addition to the specific gene
expression alterations along the cholesterol and lipid metabolism pathways, cholesterol
dependence could also be the result of insufficient precursor supply (Spens and Haggstrom,
2005).
The molecular mechanisms of host and recombinant NS0 cell lines that could be related to
the adaptation to protein-free medium are studied in this work. A quantitative study of
proteins with differential expression levels in four conditions (host NS0 cell line adapted
and non-adapted to protein-free medium, and a monoclonal antibody (Mab) transfectoma
producer NS0 adapted and non-adapted to the same protein-free medium) is reported. The
study is based on the use of the combination of two-dimensional electrophoresis and mass
spectrometry, and a novel quantitative proteomic approach, isobaric tagging for relative and
absolute quantification (iTRAQ). The metabolic study of these cell lines cultured in different
nutrient conditions is also reported. Taking into account the proteomic results and metabolic
analysis, a possible mechanism related to the adaptation of NS0 cell line to protein-free
medium is proposed.

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12 Metabolomics

4.1.1 Results: Proteomic analysis

To characterize the changes associated with the adaptation to the protein-free medium 2DE
gels of protein extracts from the cell line cultured in PFHM II with or without 1% (v/v) FBS
were compared over a pI range of 3 to 10. Following adaptation to the protein-free medium,
78 spots changed their intensity by a factor of 2 from 1200 detected spots/ treatment.
Interestingly, the majority of differentially expressed proteins decreased their expression in
cells adapted to the protein-free medium. Fifty eight proteins were characterized by
MALDI-MS and/or LC-ESI-MS-MS. The identified proteins were grouped according to their
molecular function. Four major cellular pathways seem to be involved in the adaptation to
the protein-free medium: i) carbohydrate metabolism and energy production, especially
glycolysis and the Krebs cycle, ii) protein synthesis and folding; iii) membrane transport,
and iv) cell proliferation (de la Luz et al., 2007).
In order to increase the number of proteins related with cell cycle regulation, DNA
replication and lipids synthesis we used another strategy based in the isobaric labeling and
the subcellular fractionation. iTRAQ reagent technology is a newly developed method for
relative quantification of proteins from up to four samples. It has immense potential to
improve the sensitivity and quality of mass spectrometric analysis of the proteome. We were
able to identify and quantify 575 proteins simultaneously from the four states of culture.
Among these 575 proteins, 43% were identified by a single significant peptide per protein;
the rest were identified by at least two significant peptide of the same protein. The method
used in our case to analyze the differential expression levels between two or more
conditions was the locfdr. This method is a new approach to the problem of multiple
comparisons and controls the number of false positive differentially expressed proteins
below the user-specified threshold.
The standard deviation for each peptide value is obtained from the iTracker estimates, while
in the case of proteins the variant of t-statistic suggested by Efron was used (Jung et al.,
2006) to skip the inconvenience of those proteins that were identified by a single peptide
and have standard deviation zero. In all analysis the condition locfdr 0.2 was used to select
differentially expressed proteins. This threshold is a general accepted standard in fdr
applications; it means that to consider a protein as differentially expressed the
corresponding probability of being a false positive must be below 20%.
Following this approach we have found a set of 102 differentially expressed proteins. These
proteins were classified in different functions and locations according to the KEGG database
(de la Luz et al., 2008). According with the previous results four major cellular pathways
seem to be involved in the adaptation to the protein-free medium (Figure 2).

4.1.2 Results: Kinetic and metabolic analysis

The host and recombinant NS0 cell line were culture in serum-supplemented and protein-
free medium during 140 hours. Total cell number, viable cell number and viability were
determined. The specific growth rate is different between both cells, but there was a clear
decrease when both cell lines were cultured in absence of serum (Table 3). Intracellular
metabolite concentrations were calculated during exponential growth phase. In contrast
with previous reports, we found a lost of cholesterol auxotrophy in the host and
recombinant NS0 cell line adapted to PFHM. Other metabolites such as phospholipids and

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Metabolomics and Mammalian Cell Culture 13

fructosamine involved in specific cellular processes like membrane biogenesis and

glycolipid metabolism changed their expression levels in adapted versus non-adapted cell
line. In order to check if the intracellular cholesterol concentrations increase in the adapted
cell line is a reversible process, cells were cultured in a medium supplemented with serum,
and the initial cholesterol levels were determined (de la Luz et al., 2008).

Fig. 2. Distribution of identified proteins taking in account their biological function

Table 3. Cellular growth parameters from host NS0 and recombinant NS0 cell lines adapted
and non-adapted to protein-free medium

Glycolysis is one of the most important metabolic pathways providing a source of

precursors and energy for the cell. Previous analysis by DNA microarray studies have
revealed a large number of genes involved in glycolysis, the pentose phosphate pathway
and the Krebs cycle to be down-regulated in host NS0 cell line cultured in the absence of
cholesterol (Seth et al., 2005). Ten proteins from glycolysis were found up-regulated in non
adapted NS0 cell line with respect to adapted. This result could indicate that the glycolysis
is a source of molecular precursors (cholesterol and phospholipids), especially in the
adapted cell line (Figure 3). The lactate production increase after the adaptation process
could be related with the higher lactate dehydrogenase enzyme activity (especific enzyme
activity NS0 adapted: 20.85 U*mL-1*cell-1, non-adapted: 12.31 U*mL-1*cell-1).

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14 Metabolomics

Fig. 3. Metabolic model of the adaptation of NS0 cel line to protein free-medium obtained
from proteomic and metabolites analysis. Red: sub-expressed pathways. Blue: over-
expressed pathways.

With the aim to calculate the relative rate of glycolysis and glutaminolysis, intracellular
concentration of lactate and glucose were determined in the batch culture and the
relationships between lactate production and glucose consumption (qL/qG) were calculated
(figure 4). These results indicated that the lactate produced depend of the glycolysis and the
glutaminolysis. Taken into account protemic and metabolic results we have proposed a
metabolic mechanism where the glucose is used for the precursors synthesis. On the other
hand, the cell obtain the energy from glutamine degradation.
We used the flux balance analysis (FBA) in order to compare the results obtained with an
empiric metabolic network with the experimental results. In this study we used a reported
metabolic network (Ma and Zeng, 2003) with changes in the cholesterol reactions, where the
cholesterol synthesis pathways was eliminated in NS0 non-adapted. The comparison
between adapted and non adapted metabolic network showed changes in carbohydrate and
lipid metabolism, very similar with our previous experimental results. Also we analyzed the
metabolites that have influence in cellular growth when they are not present in the medium.
Glycine, tryptophan, phenylalanine, adenine, palmitic acid, glutamic acid, methinonine and
asparagine are relatefd with the increase of cellular biomass (data not shown).

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Metabolomics and Mammalian Cell Culture 15

Fig. 4. Glucose consumption and lactate production during a batch culture of myeloma cell
line in presence and absence of serum. During the culture period, samples were taken
periodically for off-line analysis and media metabolites concentration were determined. The
relation between lactate production and glucose consumption is representative of the
cellular metabolic state, especially of the glycolysis efficiency

5. Conclusion
Data integration is not limited to flux data. Systems biology encompasses a holistic
approach to the study of biology and the objective is to simultaneously monitor all
biological processes operating as an integrated system. The use of the data obtained from
studies with different omics techniques is not simple. In addition, a single gene may code
for isoenzymes reacting with multiple metabolite substrates. The difficulty in determining
the timing of different events, that it, transcription and protein activity, also contribute to
the difficulty in integrating data. Hence in order for metabolomics to be used in systems
biology, novel strategies will need to be created. One step forward in such an integration
process is the functional assignments between protein/gene and metabolite within a system
of interest. This can be done by creating models where basic biochemical pathways are
modelled using static data (Khoo and Al-Rubeai, 2007). Second, time-dependent
concentrations of other types of components (transcriptomics and/or proteomics) will then
be incorporated followed by the reconstruction of the model with statistic data.
In contrast with previous results, changes in metabolic rates and biosynthetic machinery
with respect to the presence or not of serum in the culture medium were observed in this
study. The analysis was performed by two different ways. First, using iTRAQ reagents,
proteins with differential expression levels in two myeloma cell lines cultured in serum-
supplemented and serum-free medium were detected. These proteins belong to major
pathways related with glycolysis, protein synthesis and membrane transport. These
results are in accordance with previous results obtained using 2DE and the study of a
revertant

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16 Metabolomics

NS0 cholesterol-independent (Seth et al., 2005). Second, the determination of consumption

and production of different metabolites like glucose, lactate, cholesterol, phospholipids and
phosphorous was performed. Differences in qL/qG were found between adapted and non-
adapted cell lines, similar to the results obtained by proteomics. A significant increase was
observed in the intracellular cholesterol concentration in the adapted cell lines. However,
when these cell lines adapted to PFHM were cultured in presence of serum, the intracellular
cholesterol levels decreased down to the initial conditions, indicating a possible epigenetic
mechanism.

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 Metabolomics
Edited by Dr Ute Roessner

ISBN 978-953-51-0046-1
Hard cover, 364 pages
Publisher InTech
Published online 10, February, 2012
Published in print edition February, 2012

Metabolomics is a rapidly emerging field in life sciences, which aims to identify and quantify metabolites in a
biological system. Analytical chemistry is combined with sophisticated informatics and statistics tools to
determine and understand metabolic changes upon genetic or environmental perturbations. Together with
other 'omics analyses, such as genomics and proteomics, metabolomics plays an important role in functional
genomics and systems biology studies in any biological science. This book will provide the reader with
summaries of the state-of-the-art of technologies and methodologies, especially in the data analysis and
interpretation approaches, as well as give insights into exciting applications of metabolomics in human health
studies, safety assessments, and plant and microbial research.

How to reference
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Kathya De la Luz-Hdez (2012). Metabolomics and Mammalian Cell Culture, Metabolomics, Dr Ute Roessner
(Ed.), ISBN: 978-953-51-0046-1, InTech, Available from:
http://www.intechopen.com/books/metabolomics/metabolomics-and-mammalian-cell-cultures

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